Research is being conducted on the use of substances having specific activity, such as specific biological activity, in the cosmetics field such as skin care or hair care, but also in the dermatological and pharmaceutical fields.
Recently, the use of dsRNA and more particularly siRNA (containing 12 to 40 nucleotides) has made it possible to achieve specific activity in inhibition of the synthesis of a target protein. The molecular mechanism taking place involves double stranded RNA fragments composed of 12 to 40 nucleotides. Degradation of the target mRNA is achieved by activation of the complex known as RISC (RNA Induced Silencing Complex), which acts by fixation of the anti-sense strand of the dsRNA onto the mRNA. These double stranded RNA oligonucleotides are also known as dsRNA or else siRNA (for “short interfering” RNA); see Tuschl T., Chem. Biochem. 2001, 2, 239-245; Nykanen A et al., Cell 2001, 107, 309-321, Dorsett Y., Nature, April 2004, Vol. 3, pages 318-329; and Downward, J., BMJ 2004, 328, 1245-1248.
However, the topical application of these siRNA, which can have molecular weights of approximately 15 to 17 kD, gives rise to the problem of penetration. In fact, for the specific activity relative to the selected siRNA to be effective, it must penetrate into the target cell (such as the keratinocytes or the melanocytes, etc.) as far as its cytoplasm. As it happens, the stratum corneum, the site of the skin barrier function, is difficult to penetrate.
International Application WO 03/101376 describes cosmetic preparations comprising at least one double stranded RNA oligonucleotide. In this publication, the oligonucleotide is necessarily complexed with a cationic polymer such as PEI or chitosan, after which this complex of oligonucleotide and cationic polymer can be encapsulated in liposomes or niosomes and/or adsorbed at the surface of particles such as liposomes, niosomes, oleosomes, nanospheres and nanocapsules. Therefore, the dsRNA is necessarily complexed with a cationic polymer before being associated with a type of particle. In addition, the surfactant micelles are not described as an efficient medicinal solution for vectorization of siRNA.
In the article “Intercellular adhesion molecule-1 suppression in skin by topical delivery of anti-sense oligonucleotides,” Mehta et al. (J. Invest. Dermatol. 115, 805-812, 2000), the authors transport single stranded RNA in an emulsion containing 25% of surfactants (10% of glyceryl stearate and 15% of 40 OE stearate). Besides the fact that this article does not describe the siRNA, the proposed medicinal solution is not compatible with good tolerance. In fact, the 25% of surfactants will have the effect of destroying the barrier function of the skin, thus increasing its permeability to external elements and dramatically favoring its dehydration: results that are not sought in the fields of application envisioned by the present invention.
In the publication “Lipidic carriers of siRNA: differences in the formulation, cell uptake and delivery with plasmid DNA,” Spagnou et al. (Biochemistry, 43, 13348-56, 2004), the authors use cationic liposomes such as lipofectamine 2000 or DOPE liposomes “cationized” with cholesteryloxycarbonyl-3,7-diazanone-1,9-diamine (CDAN). For safety reasons, lipofectamine cannot be used on human skin.
Other publications (Yano Junichi et al., Clinical Cancer Research, 2004, Spagnou et al., Biochemistry, 2004, and Ma Zheng et al., Biochemical and Biophysical Research Communications, 2004) have proposed associating siRNA with cationic vesicles composed of oleyl chain lipids containing unsaturated bonds that make them very sensitive to oxidation, and phospholipids, these vesicles being intended to be used immediately after preparation, because they are not very stable in time.
International Patent Application WO 2004/046354 describes the use of siRNA for cutaneous dysfunction, preferably at the dermis level. The compositions described, such as PIT and W/O, O/W and W/O/W emulsions, are all nonionic. In that patent application, no appraisal is made of the major and essential interest of using cationic surfactants in cationic particles in order to favor penetration of the siRNA into cutaneous structures. Moreover, although the PIT can be smaller than μm size, they are necessarily nonionic, as described in International Patent WO96/28132.
International Application WO 03/106636 describes the association of a polynucleotide with a cationic surfactant such as cetyltriammonium chloride to form a complex. It is then necessary to stabilize this complex by an incubation step at a temperature of between 35 and 50° C. In a second step, it is possible to dehydrate the mixture to obtain therefrom a powder, which will then be diluted in a chosen solvent before use (injection). Once the complex has been formed, it is also possible to stabilize it by addition of an amphiphilic lipid, which can be a nonionic surfactant. This ternary association is then dehydrated to obtain a powder, which will then be diluted in an appropriate aqueous solution.
International Application WO 03/106636 does not specify that the concentration of the cationic surfactant must be above the CMC (critical micellar concentration) in order to form the complex of polynucleotide and cationic surfactant, since the complex can be formed below the CMC. It is only once this complex has been formed that the addition of another amphiphilic compound capable of forming micelles is envisioned. In this way the integration of a complex of polynucleotide and cationic surfactant into amphiphilic micelles is achieved. Thus the question of associating cationic particles with a polynucleotide is not addressed in that document.
However, there remains a need to identify a way to permit the penetration of siRNA into the skin models.